Lecture 14 - Muscle Contraction and Introduction to Enzymes

Muscle hierarchy (largest to smallest)

Muscle → bundle of muscle fibers → individual muscle fiber (multinucleated cell) → myofibril → sarcomere → thick and thin filaments

Sarcomere

• The repeating contractile unit of a myofibril, bounded by two Z disks

• contains I bands, A band, H zone, M disk, and thick and thin filaments

I band

• Light band containing only thin (actin) filaments

• spans across the Z disk

• shrinks during contraction

A band

• Dark band spanning the full length of the thick (myosin) filaments, including where they overlap thin filaments

• length stays constant during contraction

Sliding filament model of contraction

• Sarcomere shortens as thin and thick filaments slide past each other and their overlap increases

• I band and H zone shrink, A band length stays the same

• filaments themselves do not shorten

Thick filament composition

• Made of myosin

• each myosin = 6 polypeptides: 2 heavy chains (~220 kD each) + 2 pairs of light chains (essential, ELC, and regulatory, RLC)

Myosin head

• Globular N-terminal portion of the myosin heavy chain

• binds actin and hydrolyzes ATP

• each myosin molecule has two heads

Myosin tail

• C-terminal coiled-coil rod formed by the two heavy chains' α-helices (~1600 Å long: 950 Å + 650 Å segments, ~20 Å wide)

• tails bundle together to form the thick filament

Myosin Head Groups interaction

many head groups that interact with the thin chain that is composed of mostly actin. Here the micrograph depicts the complete thick filament with many myosin proteins interweaved

Thin filament composition

Mainly actin (F-actin), plus tropomyosin and troponin

G-actin vs F-actin

G-actin = globular monomer

F-actin = filamentous polymer of G-actin

Actin monomer binding and polymerization

• Each actin monomer binds ATP and a divalent cation (Ca2+)

• ATP hydrolysis —→ polymerization

Actin grooves

• Grooves along the F-actin filament that contain the myosin-binding sites

• tropomyosin lies in these grooves

Myosin head–actin binding

• Each myosin head binds in the actin groove where several binding sites lie close together

• many myosin heads attach along one thin filament at once

Tropomyosin

• Long coiled-coil protein that lies in the grooves of the actin filament

• at rest it blocks the myosin-binding sites on actin

Troponin

Three-subunit regulatory complex on the thin filament: TnC, TnI, TnT

• TnC: Troponin subunit that binds Ca2+ (the calcium sensor)

• TnI: Troponin subunit that binds actin and inhibits myosin binding (contains the "inhibitory" and "switch" regions)

• TnT: Elongated troponin subunit that binds tropomyosin at its head-to-tail junction

Titin

• Largest known polypeptide (34,350 residues) made of ~300-residue repeats

• associated with thick filaments

• acts as a spring that resists sarcomere overextension

Nebulin

Thin-filament-associated protein that sets the length of the thin filament

Dystrophin

Protein on the inner surface of the muscle plasma membrane that prevents tearing from the mechanical stress of contraction

Myosin cross-bridge cycle step 1

• ATP binds the myosin head

• actin-binding site opens and the head releases actin

Myosin cross-bridge cycle step 2

Active site closes and ATP is hydrolyzed (ADP + Pi stay bound), "cocking" the head into a high-energy, extended state like a set mousetrap

Myosin cross-bridge cycle step 3

Cocked head binds weakly to a new actin site closer to the Z disk

Myosin cross-bridge cycle step 4

Release of Pi causes the head to bind actin tightly

Myosin cross-bridge cycle step 5 (power stroke)

• Myosin head returns to its low-energy, compressed conformation and moves ~100 Å toward the Z disk along actin

• this pulls the thin filament toward the M disk and shortens the sarcomere

Myosin cross-bridge cycle step 6

• ADP is released

• myosin stays tightly bound to actin until a new ATP binds, completing the cycle

Role of Ca2+ in muscle contraction

• Ca2+ binds troponin C, causing a conformational change that shifts tropomyosin within the actin groove, exposing the myosin-binding sites

• without Ca2+, contraction cannot occur

Source of Ca2+ for contraction

The sarcoplasmic reticulum (specialized ER) releases stored Ca2+, raising cytosolic [Ca2+] from ~10^-7 M to ~10^-5 M

Relaxation and Ca2+

ATP-driven Ca2+ pumps return Ca2+ to the sarcoplasmic reticulum, lowering cytosolic Ca2+ so tropomyosin blocks myosin sites again

Four ways enzymes differ from chemical catalysts

1) Much higher reaction rates (10^6 to 10^12-fold)

2) Milder reaction conditions

3) Greater reaction specificity

4) Capacity for regulation

Do enzymes change ΔG?

No; enzymes change only the reaction rate (by lowering ΔG‡), not ΔG or the equilibrium position

Enzyme common names

Usually end in "-ase" and describe the reaction or substrate (e.g., alcohol dehydrogenase)

EC nomenclature

Enzyme Commission system giving each enzyme a 4-part number:

class.subclass.sub-subclass.serial number

so activity is unambiguous (e.g., aconitate hydratase, EC 4.2.1.3)

Catalytic power of enzymes

Understand the main idea here

Classes of Enzymes

Classes of Enzymes

Substrate specificity basis

Enzymes bind substrates through maximized noncovalent interactions:

van der Waals forces

electrostatic (ionic) bonds

hydrogen bonds

and hydrophobic interactions

Stereospecificity

Enzyme selectivity for one stereoisomer or one face/group of a molecule, because the chiral enzyme active site binds substrates asymmetrically

Aconitase stereospecificity example

• Aconitase converts citrate to isocitrate

• citrate is symmetric, yet the reaction occurs on only one of its two identical -CH2COO- arms

Prochiral center

An atom with two identical substituents (like citrate's C3) that becomes distinguishable—behaves like a chiral center—when bound to an asymmetric enzyme surface

Three-point attachment

A substrate binding an enzyme at three or more specific sites can only fit one way, so the enzyme can tell apart identical groups (e.g., pro-R vs pro-S H) and enantiomers

Chiral specificity figure

• The substrate's hydrophobic, charged, and H-bonding groups fit complementary groups in the enzyme pocket

• because binding uses multiple specific contact points, only one orientation or stereoisomer fits

Geometric specificity

• Enzymes are selective about the identity of chemical groups but are generally not specific for one molecule

• a small range of related compounds can bind or react

Chymotrypsin geometric specificity example

Chymotrypsin hydrolyzes both peptide (amide) bonds and ester bonds

Similar-shaped molecules can be toxic

• Molecules resembling natural substrates/coenzymes can bind enzymes or receptors and disrupt them

• example contrast: nicotinamide (NAD+ ring) vs tobacco nicotine

Niacin naming

Nicotinic acid was renamed niacin (by bread manufacturers) to avoid confusion with nicotine

Yeast alcohol dehydrogenase (YADH) reaction

CH3CH2OH + NAD+ ⇌ CH3CHO (acetaldehyde) + NADH + H+

NAD+ structure

• Nicotinamide + D-ribose + two phosphates + adenosine

• NADP+ has an extra phosphate on the adenosine ribose 2'-OH

NAD+ vs NADH

NAD+ = oxidized (aromatic pyridinium ring)

NADH = reduced, with a hydride added at C4 of the nicotinamide ring, giving two C4 hydrogens on opposite faces

Re-side and si-side addition

• The two faces of the nicotinamide ring

• hydride added to the re face becomes H pro-R, to the si face becomes H pro-S

Deuterium experiment with YADH

• CH3CD2OH + NAD+ → CH3CDO + NADD

• the D is transferred to one specific face of NAD+, showing stereospecific hydride transfer

YADH deuterium experiment (reverse direction)

NADD + CH3CHO gives one enantiomer of CH3CHDOH, while NADH + CH3CDO gives the opposite enantiomer, proving YADH always transfers the same (pro-R) hydrogen

YADH stereospecificity

Yeast alcohol dehydrogenase is stereospecific for the pro-R hydrogen: it adds to (forward) or removes from (reverse) the pro-R position; with the other enantiomer, D is not transferred

Pro-R vs pro-S use by dehydrogenases

• Both give identical products, but reactions with Keq for reduction >10^-12 tend to use pro-R H, while those with Keq <10^-10 tend to use pro-S H

• reason unclear

Liver ADH Leu182 mutation

• Wild-type error rate 1 in 7 billion

• Leu182→Ala raises it to 1 in 850,000 (~8000-fold increase), showing amino acid side chains maintain stereospecificity

Cofactor

• Non-protein component required for enzyme activity

• two types: metal ions and coenzymes

Coenzyme

• Small organic molecule that aids enzyme chemistry

• subdivided into cosubstrates and prosthetic groups

Prosthetic group vs cosubstrate

Prosthetic group = permanently (tightly/covalently) associated with the enzyme

cosubstrate = transiently associated, binds and leaves like a substrate (e.g., NAD+)

Holoenzyme

Catalytically active enzyme with its cofactor bound

Apoenzyme

Enzyme without its cofactor (inactive)

What enzymes CAN do alone

• Acid-base reactions

• transient covalent bonds

• charge-charge interactions

What enzymes CANNOT do alone (need coenzymes)

• Oxidation-reduction reactions

• carbon group transfers

Coenzyme regeneration

• Coenzymes must be returned to their original form to keep functioning

• e.g., glycolysis converts NAD+ to NADH, and without regenerating NAD+, glycolysis stops and death occurs

Common Coenzymes

Common Coenzymes

Vitamins and coenzymes

• Water-soluble vitamins are coenzyme precursors

• humans can't synthesize them and must get them from the diet (fat-soluble vitamins include A and D, as well as E and K)

Niacin (niacinamide) deficiency

• leads to pellagra characterized by diarrhea, dermatitis and dementia

• Pellagra was endemic is Southern United States in the early 20th century

• Niacin can be synthesized from the essential amino acid, tryptophan

• A corn diet prevalent at the time restricted the absorption of tryptophan causing a deficiency

• Treatment of corn with base could release the tryptophan (Mexican Indians treated corn with Ca(OH)2 before making tortillas!)

Two general ways to regulate enzymes

1) Control the amount/availability of the enzyme (synthesis and degradation rates)

2) Control the enzyme's catalytic activity

Control of enzyme amount

Depends on synthesis rate (transcriptional or translational control) and degradation rate

Ways catalytic activity is altered

• Positively or negatively, by conformational changes (e.g., small-molecule allosteric effectors altering the catalytic site) or covalent modifications (e.g., phosphorylation)

Allosteric effector examples (hemoglobin)

O2, CO2, and BPG binding alter hemoglobin's activity—an example of conformational regulation

Phosphorylation as regulation

• Phosphorylation of Ser residues can activate or deactivate enzymes

• usually hormonally controlled for a coordinated effect across tissues

Aspartate transcarbamoylase (ATCase)

• Catalyzes the first committed step of pyrimidine biosynthesis:

• carbamoyl phosphate + aspartate → N-carbamoyl aspartate + H2PO4-

• regulated allosterically and by feedback inhibition

ATCase kinetics curve

Sigmoidal (S-shaped) rate vs [aspartate], showing positive homotropic cooperative binding of aspartate

Homotropic vs heterotropic effects

Homotropic = substrate itself affects binding at other sites (aspartate on ATCase)

heterotropic = a different molecule acts as effector (CTP, ATP on ATCase)

CTP and ATCase

CTP is a heterotropic allosteric inhibitor of ATCase (shifts curve right)

ATP and ATCase

ATP is a heterotropic allosteric activator of ATCase (shifts curve left)

Feedback inhibition

• The end product of a metabolic pathway inhibits its own synthesis at the first committed step

• e.g., CTP inhibits ATCase (6 enzymatic steps upstream)

Why CTP regulates ATCase

• When DNA/RNA synthesis uses up CTP, CTP leaves ATCase and activity rises

• when CTP is in excess, it rebinds and inhibits

Why ATP activates ATCase

• Purines and pyrimidines are needed in equal amounts

• when ATP exceeds CTP, ATP activates ATCase until ATP and CTP levels balance

Activation energy (ΔG‡)

• The free-energy barrier between reactants and the transition state

• determines reaction rate

• it is not the same as ΔG

Transition state (X‡)

• Highest-energy, unstable arrangement along the reaction coordinate (e.g., HA---HB---HC) that all reactants must pass through

Negative ΔG and activation energy

Even if ΔG is negative (products lower in G than reactants), the reaction must still pass through the transition state, so ΔG‡ still controls the rate

Reaction intermediate

A relatively stable species (local energy minimum) between two transition states in a multistep reaction

Rate-limiting step

• The step with the highest-energy transition state

• the slowest step that controls the overall rate

Rate-limiting step example (two-step reaction A → I → P)

The step whose transition state is highest in free energy limits the rate:

in one profile A‡ is highest (first step limits)

in another, I‡ is highest (second step limits)

ΔΔG‡cat

The reduction in activation energy produced by a catalyst (uncatalyzed ΔG‡ minus catalyzed ΔG‡)

How enzymes speed reactions

Lower the activation energy (ΔG‡) without changing ΔG, so the reaction reaches equilibrium faster

Equilibrium and enzymes

Because an enzyme lowers ΔG‡ equally for the forward and reverse reactions, it speeds the approach to equilibrium without shifting the equilibrium constant